Techniques for augmentation of exogenous DNA uptake by ovine spermatozoa

Sperm mediated gene transfer can be an inexpensive and simple method in animal transgenesis; however its efficiency is poor, mainly due to the spermatozoa's lesser uptake of exogenous DNA. In the present study, the effects of lipofection and other augmentation techniques, such as sperm freezing and spermatozoa treatment with triton X100 and DMSO, on exogenous DNA uptake by sheep spermatozoa and motility of sperms with plasmid uptake were evaluated. In the first experiment, ram sperms were incubated with a complex of rhodamine labeled plasmid [p-EGFP] and Lipofectamine 2000TM. In the second, spermatozoa were treated with Triton X-100TM or DMSO or were frozen without cryoprotectant. The results indicated that there was no significant difference [P<0.05] in the transfection rates and in the uptake intensity of lipofected sperms with 300 and 600 ng of plasmid in comparison with control group, i.e. transfected without lipofectamine. Furthermore, lipofection could not improve sperm motility during true plasmid uptake. Almost all of triton X100 treated and frozen-thawed spermatozoa had absorbed foreign DNA, though all were immotile. In spermatozoa treated with 0.1% DMSO, plasmid absorption rate [69.40%] was significantly higher [P<0.05] than untreated spermatozoa [57.80%], but sperm motility was not significantly different from control group. In conclusion, lipofectamine® 2000 could neither improve transfection rate, nor support motility in transfected sperms. The methods inducing membrane disruption like, freeze-thaw and triton X100 treatment, can be used in ICSI-sperm mediated gene transfer without the need for sperm selection, provided that they cause no damage to sperm nucleus

[Transfection of ram spermatozoa with pEGFP carrying human lysozyme gene]

As we all know, sperm has the capacity to take up foreign DNA, therefore, sperm mediated gen transfer can be an inexpensive and simple method in animal transgenesis in various species. However, there is not sufficient evidence of DNA uptake by ovine spermatozoa. The purpose of the present study was to examine the uptake of human lysozyme gene contained plasmid [pEGFP-IRES-hLys] by ovin spermatozoa. In the first experiment, semen was prepared from three ram [each ram two times] by electrical method. After removal of seminal plasma, 1x106 spermatozoa were incubated with rhodamin-labled pEGFP-IRES-hLys in TCM199 for 15, 30, 60 and 120 minutes and then observed for motility, uptake percent and uptake intensity by florescent microscopy. Also after 60 minutes incubation sperms were treated by DNaseI to assay adsorption or uptake of pEGFP-IRES-hLys. In the second experiment, washed and unwashed sperms were incubated with rhodamin-labled pEGFP-IRES-hLys in TCM199 for 30 and 60 minutes to evaluate the effect of presence seminal plasma on sperm uptake and motility. The findings showed that increasing incubation time increased number/percentage of spermatozoa carrying exogenous DNA and its intensity. But this different was significant only up to 30 minutes. We found that 60.16% of the cells were bound to DNA after 120 minutes incubation. Incubation with exogenous DNA induced a slightly decrease in sperm total and progressive motility. But no post acrosom uptaked sperm was motile. After treatment with DNaseI, strong florescent emission from post acrosom indicated absorption of pEGFP-IRES-hLys by spermatozoa. Presence of seminal plasma induced a slightly decrease in percent of DNA absorbed spermatozoa and absorption intensity, but did not inhibit completely. Ram spermatozoa showed a high capacity to bind DNA quickly and reach a maximum after 30 min. However, no sperm with real uptake [post acrosomal] was motile. Incubation with lower DNA concentration and/or shorter time may be helpful

Relationship between DNA polymorphisms at the BCL11A and HBS1L-MYB loci in beta-Thalassemia patients with increased fetal hemoglobin levels

High fetal hemoglobin [HbF] levels have a major impact on the hemoglobin disorders, i.e. beta -Thalassemia. Increased HbF production ameliorates the disease severity. Three loci-HBS1L-MYB intergenic region on chromosome 6q23, BCL11A on chromosome 2pl6, and the chi-globin gene on chromosome 11 account for up to 50% of the variations in HbF levels in patients with sickle cell anemia, thalassemia and healthy adults. In the present study, we evaluated the relationship between some polymorphisms on HBS1L-MYB BCL11A loci and increased HbF levels in thalassemia patients and normal subjects. In this case-control study, three common polymorphisms among 50 beta-thalassemia patients with increased HbF and 47 healthy individuals with normal HbF by using PCR-RFLP were genotyped: rs4895441, rsl 1886868, and rs28384513. Enzymatic digestion was performed by Rsal, MboII, and BstXI, respectively. Correlations with high levels of HbF were performed with a Chi-square test by using SPSS 16 and SNP analyzer2. Mutant allelic frequencies were 0.245, 0.521 and 0.309 in healthy and 0.3, 0.52 and 0.28 in patient for rs4895441, rsl 1886868 and rs28384513, respectively. Significant relationship was not observed among three polymorphisms studied in healthy volunteers and beta-Thalassemia major patients with increased HbF levels and P-value allelic and genotypic was higher than 0.05 at three SNPs. In spite of previous reports, evaluation of polymorphisms at the BCL11A and HBS1L-MYB loci in this study did not show up a significant correlation with increased HbF. Other polymorphisms might have a role in increasing HbF in our population